Age-differential sexual dimorphisms in CHD8-S62X-mutant mouse synapses and transcriptomes

Chd8+/N2373K mice with a human C-terminal-truncating mutation (N2373K) display autistic-like behaviors in juvenile and adult males but not in females. In contrast, Chd8+/S62X mice with a human N-terminal-truncating mutation (S62X) display behavioral deficits in juvenile males (not females) and adult males and females, indicative of age-differential sexually dimorphic behaviors. Excitatory synaptic transmission is suppressed and enhanced in male and female Chd8+/S62X juveniles, respectively, but similarly enhanced in adult male and female mutants. ASD-like transcriptomic changes are stronger in newborn and juvenile (but not adult) Chd8+/S62X males but in newborn and adult (not juvenile) Chd8+/S62X females. These results point to age-differential sexual dimorphisms in Chd8+/S62X mice at synaptic and transcriptomic levels, in addition to the behavioral level.


Introduction
Mutations of CHD8, encoding a chromatin remodeler, have been extensively associated with autism spectrum disorders (ASD; Bernier et al., 2014;Barnard et al., 2015;Dingemans et al., 2022;Zhou et al., 2022). Human individuals carrying CHD8 mutations show a strong male-female ratio (~85:15; Stessman et al., 2017), suggesting that CHD8 could be one of the ideal targets for studying the mechanisms underlying male-female differences in ASD.
Chd8 +/N2373K mice that carry a human mutation that leads to a C-terminal protein truncation (N2373K; O'Roak et al., 2012;Merner et al., 2016) display male-preponderant behavioral deficits as juveniles and adults. In contrast, Chd8 +/S62X mice expressing CHD8-S62X proteins with N-terminal truncation (O'Roak et al., 2012) display behavioral deficits in juvenile males (not females) and adult Frontiers in Molecular Neuroscience 02 frontiersin.org males and females , indicative of age-differential sexually dimorphic behaviors. Here, we characterized and compared synaptic and transcriptomic phenotypes in male and female Chd8 +/S62X mice at juvenile and adult stages, which led us to find age-differential sexual dimorphisms at synaptic and transcriptomic levels additional to the behavioral level.

Materials and methods Animals
Chd8 +/S62X mice have been recently reported . Mice were maintained at the Korea Advanced Institute of Science and Technology (KAIST) mouse facility (12-h light-dark cycle).

RNA-Seq analysis
Three mice aged P0, P25, and P80 were used for each group (heterozygous, wildtype, male, female). Brains were quickly dissected and deep-freezed in RNAlater solution (Ambion) to stabilize RNAs. RNA extraction, library preparation, cluster generation, and sequencing were conducted by Macrogen. Sequencing was performed with an average read depth of 70 to 90 million reads at paired-ends (2 × 101 bp) using an Illumina HiSeq 4000 (Illumina) via Macrogen Inc. Transcript abundance was estimated in pseudo-mapping-based mode for the Mus musculus genome (GRCm38) using Salmon (v1.1.0; Patro et al., 2017). Differential gene expression analysis was performed using R/Bioconductor DEseq2 (v1.26.0; Love et al., 2014) by importing the estimated abundance data into R (v.3.5.3) using the tximport (Soneson et al., 2015) package. The p-values were adjusted for multiple testing with the Benjamini-Hochberg correction. A threshold for differentially expressed genes was used as a gene with an adjusted p-value of less than 0.05. We did not attempt RT-qPCR validation of RNA-Seq results considering that RNA-Seq results are usually well correlated with RT-qPCR results and that our study extracts most of the biological functions through GSEA (not DEG analysis), which uses a large number of genes with small changes (Subramanian et al., 2005) that are difficult to validate by RT-qPCR.

Gene set enrichment analysis
Fold changes and adjusted p-values from differential gene expression (DEG) analysis were used to perform Gene Set Enrichment Analysis (GSEA; Subramanian et al., 2005). GSEA allows us to capture if the expressions of genes in a specific gene set are changed in a consistent direction, although each might not be significant enough to be counted as differentially expressed genes. The GSEA software (gsea2-2.2.4.jar; http://software.broadinstitute.org/gsea) was used to obtain enrichment scores. All expressed genes were ranked by the sign of fold change multiplied by -log10 (p-value). 1,000 permutations and a classic scoring scheme were used, as recommended by the program. Gene sets used in this study included those from the Molecular Signature Database (MSigDB v7.0; http://software.broadinstitute.org/gsea/msigdb) and ASD-related gene sets from previous studies (gene sets that are either upregulated or downregulated in ASD and ASD-risk gene sets; see the Results section below for details; Voineagu et al., 2011;Iossifov et al., 2014;Werling et al., 2016;Yang et al., 2018). GSEA results were visualized using EnrichmentMap App in Cytoscape 3.8.0. The RNA-Seq results are deposited in the NCBI GEO (National Center for Biotechnology Information, Gene Expression Omnibus) repository as GSE167053.

Statistics
Outlying data were excluded using ROUT test (Q = 1%). For malefemale comparisons, two-way ANOVA (sex and genotype factors) with assumed normality was used. Graphpad Prism 9 and SigmaPlot 12.0 programs were used for statistical analyses. Statistical details are shown in Supplementary Table S1.

Increased excitatory synaptic transmission in adult Chd8 +/S62X males and females
To determine whether the similarly altered behaviors in adult Chd8 +/S62X males and females  accompany synaptic dysfunctions, Frontiers in Molecular Neuroscience 03 frontiersin.org we measured synaptic transmissions in the adult hippocampus, a brain region associated with ASD (Schumann et al., 2004). Adult (13-18 weeks) male and female Chd8 +/S62X mice showed significant increases in miniature excitatory postsynaptic currents (mEPSCs) in CA1 hippocampal neurons compared with WT mice, as supported by genotype differences in mEPSC frequency and amplitude in two-way ANOVA analysis ( Figure 1A; Supplementary Table S1). However, an additional Mann-Whitney U-test performed, based on the insignificant sex x genotype interaction in the two-way ANOVA, indicated a genotype difference in females but not in males in the frequency and no genotype difference in males or females in the amplitude. In contrast to these excitatory synaptic changes, there were no alterations in miniature inhibitory postsynaptic currents (mIPSCs) in Chd8 +/S62X males or females compared with WT mice, although females showed greater mIPSC frequency and amplitude compared with males ( Figure 1B).
The excitatory synaptic differences, however, became insignificant when spontaneous excitatory postsynaptic currents (sEPSCs) were recorded by omitting the action-potential blocker tetrodotoxin in recording solutions to allow network activity ( Figure 1C). Spontaneous inhibitory postsynaptic currents (sIPSCs) were also not different between genotypes ( Figure 1D).
These results collectively suggest that the CHD8-S62X mutation enhances excitatory, but not inhibitory, synaptic transmission in adult male and female mutant mice, and that network activity compensates for the mutation-induced excitatory synaptic changes. Increased excitatory synaptic transmission in adult Chd8+/S62X males and females. Opposite changes in excitatory synaptic transmission in juvenile Chd8 +/S62X males and females Given the stronger mother-seeking/attachment behavior in juvenile male Chd8 +/S62X mice , we next tested whether juvenile (postnatal day [P] 22-28) Chd8 +/S62X males and females show any differential changes in hippocampal mEPSCs or mIPSCs.
Intriguingly, juvenile male and female Chd8 +/S62X mice showed opposite changes in mEPSCs, but not mIPSCs, compared with WT mice, with the frequency of mEPSCs decreasing in males but increasing in females; the amplitudes of mEPSCs were unaffected in either sex (Figures 2A,B). In addition, allowing network activity during recordings by omitting tetrodotoxin did not affect the oppositely changed mEPSC frequencies observed in juvenile male and female Chd8 +/S62X mice ( Figures 2C,D), in contrast to the abovementioned compensatory effects of network activity on mEPSCs in adult Chd8 +/S62X mice. However, a moderate decrease in Frontiers in Molecular Neuroscience 05 frontiersin.org sIPSC amplitude (not frequency) was observed in male but not female Chd8 +/S62X mice in the presence of network activity ( Figure 2D). Therefore, the CHD8-S62X mutation results in the opposite, or sexually dimorphic, changes in excitatory, but not inhibitory, synaptic transmissions that are resistant to network correction in the juvenile mice, which contrasts with the sensitivity of the excitatory synaptic change to network correction in adult mutant mice.
Age-differential ASD-like transcriptomic changes in male and female Chd8 +/S62X mice The results recently reported  and mentioned above indicate largely similar synaptic and behavioral phenotypes in adult male and female mutant mice but sexually dimorphic synaptic changes and male-preponderant behavioral deficits in juvenile mutant males and females. To better understand the mechanisms underlying these results, we performed a longitudinal analysis of transcriptomic changes in the whole brain of P0 (newborn), P25 (juvenile), and P80 (adult) Chd8 +/S62X males and females (see Supplementary Table S2 for raw RNA-Seq data).
Analyses of the RNA-Seq results with a focus on differentially expressed genes (DEGs) did not reveal detectable changes in biological functions, likely because of the small numbers of DEGs in P0, P25, and P80 Chd8 +/S62X males and females (Figures 3A-F; see  Supplementary Table S3 for the complete list of DEGs). We thus performed a gene set enrichment analysis (GSEA), a method optimized to identify biological functions that depend on a large number of genes Age-differential ASD-like transcriptomic changes in adult Chd8+/S62X males and females. (A) GSEA of P0, P25, and P80 male and female HT/WT transcripts for ASD-related/risk gene sets, including those upregulated in ASD (DEG_Up_Voineagu and Co-Exp_Up_M16_Voineagu) and downregulated in ASD (DEG_Down_Voineagu and Co-Exp_Down_M12_Voineagu) as well as ASD-risk gene sets that are likely downregulated in ASD (SFARI [all], SFARI [high confidence], FMRP targets, DeNovoMis, DeNovoVariants, and AutismKB). (see Supplementary Tables S5-S7 for full results). (n = 3 mice for P0/25/80, male/ female, and WT/HT mice, FDR < 0.05). (B) GSEA of P0, P25, and P80 male and female HT/WT transcripts for cell-type-specific gene sets related to neurons (cortical layers and GABA sub-types). (n = 3 mice for P0/25/80, male/female, and WT/HT mice, FDR < 0.05). (C) GSEA of P0, P25, and P80 male and female HT/WT transcripts for cell-type-specific gene sets related to glial cells (oligodendrocytes, astrocytes, and microglia; n = 3 mice for P0/25/80, male/female, and WT/HT mice, FDR < 0.05).
with small, but coordinated, changes rather than by a small number of genes with large changes above artificial cutoffs. 1 GSEA results revealed that, at P0, both male and female Chd8 +/S62X mice showed transcriptomic changes similar to those that occur in ASD (hereafter termed ' ASD-like pattern'). Specifically, P0 male HT/WT transcripts-the complete set of transcripts differentially expressed between heterozygous Chd8 +/S62X mice and WT mice (ranked by foldchange signs x -log10 [p-value])-were positively enriched for ASD-related gene sets (summarized in Supplementary Tables S4, S5) that are upregulated in ASD (DEG_Up_Voineagu and Co_Exp_Up_ Voineagu;Voineagu et al., 2011;Werling et al., 2016), although not negatively enriched for ASD-downregulated gene sets (DEG_Down_ Voineagu and Co_Exp_Down_Voineagu; Figure 4A). In addition, P0 male HT/WT transcripts showed negative enrichments for ASD-risk gene sets such as SFARI genes 2 and FMRP targets (Werling et al., 2016) but not for DeNovoMis (protein-disrupting or missense rare de novo variants; Iossifov et al., 2014;Werling et al., 2016), DeNovoVariants (protein-disrupting rare de novo variants; Iossifov et al., 2014;Werling et al., 2016), or AutismKB (Autism KnowledgeBase; Yang et al., 2018; Figure 4A); these gene sets are thought to be downregulated by ASD-related deletion, non-sense, missense, frame-shift, and/or splice-site mutations. P0 female HT/WT transcripts showed positive and negative enrichments in ASD-related and ASD-risk gene sets. This is similar to the changes observed in P0-male HT/WT transcripts, which indicates comparable transcriptomic changes in P0 mutant males and females. In contrast, P25 male and female HT/WT transcripts showed distinct enrichment patterns. P25 males showed an "ASD-like" pattern similar to that observed in ASD as well as P0 males and females, whereas P25 females showed a pattern that is largely unrelated to ASD, with minimal enrichments for ASD-related/risk gene sets ( Figure 4A; Supplementary Table S6).
Intriguingly, at P80, male HT/WT transcripts showed minimal enrichment for ASD-related/risk gene sets, whereas female HT/WT transcripts displayed a strong ASD-like pattern ( Figure 4A; Supplementary Table S7). These results indicate that mutant male transcriptomes show strong ASD-like, monophasic patterns at ~P0 and P25 but not at P80, whereas mutant female transcriptomes show strong ASD-like patterns at ~P0 and P80 (a biphasic pattern), with a weak or no ASD-like pattern at ~P25.
Previous studies have also reported that ASD is associated with celltype-specific transcriptomic changes, including decreased gene expression in neurons and oligodendrocytes and increased gene expression in astrocytes and microglia (Voineagu et al., 2011;Werling et al., 2016), although alternative splicing of these genes controlled by proteins, including RBFOX1, can provide a distinct layer of regulation (Irimia et al., 2014;Parikshak et al., 2016;Quesnel-Vallieres et al., 2019). Tests of Chd8 +/S62X transcriptomes for the enrichment of cell-typespecific gene sets (Albright and Gonzalez-Scarano, 2004;Cahoy et al., 2008;Kang et al., 2011;Zeisel et al., 2015;Werling et al., 2016;Velmeshev et al., 2019Velmeshev et al., , 2020 showed that both male and female P0 HT/WT transcriptomes were negatively enriched for excitatory neuron-related gene sets but positively enriched for astrocyte-and microglia-related gene sets ( Figures 4B,C), consistent with "ASD-like" patterns. However, mixed positive and negative enrichments could also be observed for some gene sets associated with inhibitory neurons and oligodendrocytes, standing in between the ASD-like pattern and an opposite change (hereafter termed "reverse-ASD-like" pattern).
P25 male HT/WT transcripts maintained this tendency toward an ASD-like pattern, with the negative enrichment for inhibitory neurons getting stronger, but P25 female HT/WT transcripts showed a pattern opposite that of the ASD-like pattern (i.e., nearly undetectable neuronal enrichments, upregulated oligodendrocyte genes, and downregulated astrocyte and microglial genes; Figures 4B,C). At P80, male HT/WT transcripts showed enrichment patterns that were partly ASD-like (moderately downregulated neuronal genes and downregulated oligodendrocyte genes) and reverse-ASD (downregulated astrocyte and microglia genes), whereas female HT/WT transcripts showed a strong ASD-like pattern (downregulated neuronal and oligodendrocyte genes and upregulated astrocyte and microglia genes).
These results collectively suggest that male HT/WT transcripts show monophasic ASD-like transcriptomic changes that peak at ~P0 and P25, whereas female HT/WT transcripts show biphasic transcriptomic changes peaking at ~P0 and P80.
In the cellular component (CC) domain, P0 male HT/WT transcripts were positively enriched for extracellular matrix-and chromatin-related gene sets, as shown by the top-five gene-set list ( Figure 5A; see Supplementary Tables S5-S7 for full GSEA results [GSEA-CC]) and the clusters of enriched gene sets revealed by the EnrichmentMap Cytoscape App (Isserlin et al., 2014; Figure 5B). P0 male HT/WT transcripts were also negatively enriched for synapse-, spliceosome-, ribosome-, mitochondria-, and chromatin remodelingrelated gene sets, as shown by top-five and clustered gene sets ( Figures 5A,B). P0-female-HT/WT transcripts showed similar positive and negative enrichment patterns.
At P25, male HT/WT transcripts were negatively enriched for synapse-, ribosome-, and mitochondria-related gene sets ( Figures 5A,B), a pattern with some similarity to that observed in P0 males. In P25 females, however, HT/WT transcripts were positively enriched for ribosome-and mitochondria-related gene sets ( Figures 5A,B), a pattern largely opposite that observed in P25 males.
At P80, male HT/WT transcripts showed minimal enrichment patterns (positive or negative), whereas female HT/WT transcripts showed strong negative enrichments for synapse-related gene sets and positive enrichments for ribosome/mitochondria-related gene sets ( Figures 5A,B).
Tests of gene sets in biological process and molecular function domains (C5 database) revealed enrichment patterns that are largely similar to those observed for CC-domain gene sets, although MF-domain gene sets tended to show weaker enrichments ( Figure 6A,B; Supplementary Figure S1; Supplementary Tables S5-S7).
These results collectively suggest that synapse genes are downregulated at P0 and P25 in mutant males and at P0 and P80 in mutant females. Notably, downregulations of synapse genes in P25 males and P80 females were strongly correlated with the ASD-like transcriptomic patterns (Voineagu et al., 2011;Werling et al., 2016) observed in these animals.

Discussion
We characterized synaptic and transcriptomic phenotypes of Chd8 +/ S62X males and females at multiple postnatal stages and found age-differential sexual dimorphism at both synaptic and transcriptomic levels.
Our previous study Chd8 +/S62X mice revealed that behavioral deficits are observed in male but not female juveniles, whereas adult mutant males and females display similar behavioral deficits . This age-differential sexual dimorphism in the behaviors of Chd8 +/S62X mice differs from the persistent male-preponderant behavioral deficits in Chd8 +/N2373X pups, juveniles, and adults (Jung et al., 2018). What might be the mechanisms underlying the age-differential behavioral deficits in Chd8 +/S62X males and females?
Our results indicated decreased excitatory synaptic transmission in male Chd8 +/S62X juveniles. This change was correlated with both ASD-like transcriptomic changes and autistic-like behaviors (enhanced mother seeking/attachment), suggesting that the decreased excitatory transmission contributes to the behavioral deficits. In contrast, female juvenile mutant mice showed increased excitatory synaptic transmission that correlated with considerably weakened ASD-like transcriptomic patterns (relative to newborn-stage patterns) and largely normal behaviors. Therefore, excitatory synaptic suppression likely underlies the male-preponderant behavioral deficits in juvenile mutant mice. This hypothesis is in line with the synaptic dysfunction frequently implicated in ASD (Bourgeron, 2015), active synaptic development at juvenile Frontiers in Molecular Neuroscience 08 frontiersin.org stages (i.e., 2-3 postnatal weeks; Sheng and Sala, 2001), and neuronal/ synaptic excitation/inhibition imbalances implicated in ASD (Nelson and Valakh, 2015;Lee et al., 2017). Notably, overproduction of inhibitory neurons, which would also induce a decrease in the excitation/inhibition ratio, similar to our results (glutamate synaptic suppression), was observed in human ASD-related cortical organoids (Mariani et al., 2015), suggesting that distinct mechanisms in mice and humans may underlie a similar excitation/inhibition imbalance. At adult stages, Chd8 +/S62X males and females show strong and shared autistic-like behavioral deficits (self-grooming and anxiety-like behaviors; Lee et al., 2022). These mice also exhibit shared increases in excitatory synaptic transmission (mEPSC results). These strong correlations suggest that the increased excitatory synaptic transmission may underlie autistic-like behaviors in mutant males and females at adult stages. While displaying similar increases in excitatory synaptic transmission as adults, Chd8 +/S62X males and females seem to undergo distinct synaptic and transcriptomic changes across the juvenile-to-adult temporal axis. Specifically, Chd8 +/S62X males show decreased excitatory synaptic transmission as juveniles but increased excitatory transmission as adults. This age-dependent reversal could reflect the consequence of excessive compensatory changes to normalize the decreased juvenile excitatory synaptic transmission. Such compensatory changes and Biological functions differentially altered in male and female Chd8 +/S62X transcriptomes. (A) GSEA of P0, P25, and P80 male and female HT/WT transcripts for gene sets in the cellular components (CC) domain of the C5 database (http://software.broadinstitute.org/gsea). Top-five gene sets positively (red circles) or negatively (blue circles) enriched in male or female transcripts are listed, together with the same gene sets in the other sex and their enrichment levels (see Supplementary Tables S5-S7 for full results). (n = 3 mice for P0/P25/P80 male/female mice, FDR (false discovery rate) < 0.05; NES, normalized enrichment score). (B) Integration and visualization of enriched genes in larger clusters using Cytoscape EnrichmentMap. (n = 3 mice for P0/25/80 male/female mice; node cutoff, FDR < 0.05 and p < 0.001; edge cutoff, overlap coefficient > 0.5).
Frontiers in Molecular Neuroscience 09 frontiersin.org synaptic normalization may induce the substantial weakening of ASD-like transcriptomic patterns that occurs between juvenile and adult stages, although these compensations appear to be not sufficient to rescue the behavioral deficits.
Adult female Chd8 +/S62X mice, unlike adult males, show excitatory synaptic transmission that continues to be increased across juvenile and adult stages. However, the weakened ASD-like transcriptomic patterns in juvenile female mutant mice, relative to Transcriptomic changes in Chd8 +/S62X males and females for gene sets in the biological process domain. (A) GSEA of P0, P25, and P80 male and female HT/ WT transcripts for gene sets in the biological process (BP) domain of the C5 database (http://software.broadinstitute.org/gsea). Top-five gene sets positively or negatively enriched in male or female transcripts are listed, together with the same gene sets in the other sex and their enrichment levels (see Supplementary Tables S5-S7 for complete results). (n = 3 mice for P0/P25/P80 male/female mice, FDR (false discovery rate) < 0.05; NES, normalized enrichment score). (B) Integration and visualization of enriched genes in larger clusters using Cytoscape EnrichmentMap. (n = 3 mice for P0/25/80 male/ female mice; node cutoff, FDR < 0.05 and p < 0.001; edge cutoff, overlap coefficient > 0.5).
newborn mice, become strong again in adult female mutant mice in a biphasic manner, similar to the ASD-like transcriptomic patterns in newborn mice. It is possible that the increased excitatory synaptic transmission, which likely rescues the behavioral deficits in juvenile mutant females, may no longer be able to protect adult females from developing behavioral deficits. Notably, in adult mutant females, the increased excitatory synaptic transmission in adult mutant females is sensitive to network compensation, as supported by the adult sEPSC results, unlike the increased excitatory synaptic transmission in juvenile mutant females that is resistant to network compensation (juvenile sEPSC results). This may represent acute adult-onset compensatory changes aiming to normalize excitatory synaptic transmission, which may be mediated by the strongly suppressed synaptic gene expressions, although this might induce secondary synapse-related changes that aggravate brain functions and behaviors.
The data collected from Chd8 +/S62X mice in the present and previous studies are limited by the lack of causal relationships between the observed molecular, synaptic, and behavioral changes. Although additional details remain to be determined, our data may provide some baseline information given that little is known about (1) whether the strong male preponderance in autistic individuals with CHD8 mutations could be recapitulated in mice, (2) whether different CHD8 mutations lead to heterogeneous male-female differences in mice in a spatiotemporally differential manner, and (3) what mechanisms underlie the CHD8-related sexual dimorphism and the general sexual dimorphism widespread in ASD and neurodevelopmental and psychiatric disorders.
In summary, our results provide evidence suggesting that the CHD8-S62X mutation derived from an autistic individual leads to age-differential and sexually dimorphic synaptic and transcriptomic changes in mice.

Data availability statement
The RNA-Seq results are deposited in the NCBI GEO (National Center for Biotechnology Information, Gene Expression Omnibus) repository as GSE167053..

Ethics statement
The animal study was reviewed and approved by Committee on Animal Research at KAIST.